Self-sealed fluidic channels for a nanopore array

ABSTRACT

A method of forming a nanopore array includes patterning a front layer of a substrate to form front trenches, the substrate including a buried layer disposed between the front layer and a back layer; depositing a membrane layer over the patterned front layer and in the front trenches; patterning the back layer and the buried layer to form back trenches, the back trenches being aligned with the front trenches; forming a plurality of nanopores through the membrane layer; depositing a sacrificial material in the front trenches and the back trenches; depositing front and back insulating layers over the sacrificial material; and heating the sacrificial material to a decomposition temperature of the sacrificial material to remove the sacrificial material and form pairs of front and back channels, wherein the front channel of each channel pair is connected to the back channel of its respective channel pair by an individual nanopore.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/092,424, filed Apr. 22, 2011, the disclosure of which is incorporatedby reference herein in its entirety.

BACKGROUND

This disclosure relates generally to the field of sensors for sequencingdeoxyribonucleic acid (DNA) and proteins.

DESCRIPTION OF RELATED ART

Mapping the sequence of bases of a DNA strand is of great importance inlife sciences. Current DNA sequencing technologies based on sequencingby synthesis cost more than $300,000 per human genome, and there isgreat demand to lower the cost to less than $1000 per human genome forpurposes such as personalized medicine and preventive Medicare. Since asingle base is about 0.7 nanometers (nm) long when the DNA strand isstretched, it is important that a sensor for sequencing have a spatialresolution of about 1 nm or less. Fabricating a sensor with a spatialresolution in this range, however, is challenging. Another area of greatimportance in life sciences is detection of proteins and viruses. Forprotein and viruses detection, a biomolecule sensor that comprises afield effect transistor (FET) may be used. However, a disadvantage ofmany FET-based sensors is that a sensing surface of the sensor must becovered with a biological coating that specifically binds thebiomolecules to be detected. Applying the appropriate coating may belabor intensive and expensive. Further, the FET sensor may only be usedto detect the particular biomolecules that bind with the coating,limiting the usefulness of the sensor.

A FET sensor may comprise highly doped source and drain regions formedby ion implantation, and followed by high temperature annealing (e.g.,about 1000° C.). Though this method is standard for forming source anddrain regions in longer channel (greater than 10 nm) FET devices, ionimplantation and anneal may pose a problem for fabrication of relativelyshort FET channels (less than about 5 nm) required for high sensitivityFET devices, as ion implantation and high temperature activationannealing produce dopant density profiles in the source/drain regionsthat may extend several nanometers into the channel region of the FET.Consequently, the sensitivity of a FET sensor formed in this manner maybe degraded, making the FET sensor inappropriate for use for sequencingDNA.

A nanopore sensor has been proposed as a potential approach for DNAsequencing at cost less than $1000 per human genome, and may be used asan alternative to a FET sensor for biomolecule sensing. Because DNAmolecules have a relatively high negative charge, a DNA molecule may beelectrically driven from a first fluidic reservoir to a second fluidicreservoir through a nanopore that has a diameter on the order of a fewnanometers. FIG. 1 shows a nanopore sensor system 100 including a singlenanopore 103 according to the prior art. The nanopore 103 is formedthrough a membrane 101. The membrane 101 partitions a fluidic reservoir104 into two parts: top reservoir 105 and bottom reservoir 106. Thefluidic reservoir 104 and the nanopore 103 are then filled with a fluid107, which may be an ionic buffer, which contains biomolecules such asDNA molecule 108. The DNA molecule 108 is translocated through nanopore103 by an electrical voltage bias 109 that is applied across thenanopore 103 via two electrochemical electrodes 110 and 111, which aredipped in the top fluid reservoir 105 and the bottom fluid reservoir106, respectively. As the DNA molecule 108 moves through the nanopore103, the DNA molecule 108 is sequenced via ionic current through thenanopore or other optical/electrical sensors integrated near thenanopore 103. Besides DNA sequencing, such nanopore sensors may performrelatively rapid analysis of biomolecules, such as DNA, RNA, andproteins, in addition to providing information regarding biomoleculeinteractions. Nanopore DNA sequencing is a real-time single moleculemethod, without the need of DNA amplification or chemically modifyingthe DNA, and offers a relatively lowest cost method for DNA sequencing.

BRIEF SUMMARY

In one aspect, a method of forming a nanopore array includes patterninga front layer of a substrate to form front trenches, the substrateincluding a buried layer disposed between the front layer and a backlayer; depositing a membrane layer over the patterned front layer and inthe front trenches; patterning the back layer and the buried layer toform back trenches, the back trenches being aligned with the fronttrenches; forming a plurality of nanopores of the nanopore array throughthe membrane layer; depositing a sacrificial material in the fronttrenches and the back trenches; depositing front and back insulatinglayers over the sacrificial material; and heating the sacrificialmaterial to a decomposition temperature of the sacrificial material toremove the sacrificial material and form pairs of front and backchannels, wherein the front channel of each channel pair is connected tothe back channel of its respective channel pair by an individualnanopore.

In another aspect, a nanopore array includes a plurality of front andback channel pairs located in a substrate, wherein the front channel ofeach channel pair is connected to the back channel of its respectivechannel pair by a single nanopore of a plurality of nanopores, whereinthe nanopores are formed through a membrane layer that is locatedbetween the front channels and the back channels; wherein the pluralityof front channels of the plurality of front and back channel pairs arelocated in a patterned front layer of the substrate and bounded on topby a front insulating layer; wherein the membrane layer is located overthe patterned front layer and in a bottom portion of the plurality offront channels; and wherein the plurality of back channels of theplurality of front and back channel pairs are located in a patternedburied layer and a patterned back layer of the substrate and bounded atthe bottom by a back insulating layer.

Additional features are realized through the techniques of the presentexemplary embodiment. Other embodiments are described in detail hereinand are considered a part of what is claimed. For a better understandingof the features of the exemplary embodiment, refer to the descriptionand to the drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Referring now to the drawings wherein like elements are numbered alikein the several FIGURES:

FIG. 1 is a schematic diagram illustrating a cross section of anembodiment of a nanopore sensor system for DNA sequencing according tothe prior art.

FIG. 2 is a flowchart illustrating an embodiment of a method of formingself-sealed fluidic channels for a nanopore array.

FIG. 3 is a schematic diagram illustrating a cross section of anembodiment of a substrate.

FIG. 4 is a schematic diagram illustrating a cross section of thesubstrate of FIG. 3 after patterning the front layer of the substrate toform front trenches.

FIG. 5 is a schematic diagram illustrating a cross section of the deviceof FIG. 4 after depositing of a membrane layer over the patterned frontlayer and the front trenches.

FIG. 6 is a schematic diagram illustrating a cross section of the deviceof FIG. 5 after formation of a mechanical support layer over themembrane layer.

FIG. 7 is a schematic diagram illustrating a cross section of the deviceof FIG. 6 after opening windows in the mechanical support layer.

FIG. 8 is a schematic diagram illustrating a cross section of the deviceof FIG. 7 after patterning the back layer and the buried layer of thesubstrate to form back trenches.

FIG. 9 is a schematic diagram illustrating a cross section of the deviceof FIG. 8 after forming nanopores in the membrane layer.

FIG. 10 is a schematic diagram illustrating a cross section of thedevice of FIG. 9 after deposition of a thermally decomposablesacrificial material in the front and back trenches.

FIG. 11 is a schematic diagram illustrating a cross section of thedevice of FIG. 10 after deposition of an insulating material over thefront and back sacrificial material.

FIG. 12 is a schematic diagram illustrating a cross section of thedevice of FIG. 11 after removal of the sacrificial material.

FIG. 13 is a schematic diagram illustrating a top view of a nanoporearray including self-sealed fluidic channels formed by the method ofFIG. 2.

FIG. 14 is a schematic diagram illustrating a cross section of onenanopore of the nanopore array of FIG. 13.

FIGS. 15 a-b illustrates thermally decomposable polymers that may beused for the sacrificial material in various embodiments.

DETAILED DESCRIPTION

Embodiments of self-sealed fluidic channels for a nanopore array andmethods of making self-sealed channels for a nanopore array areprovided, with exemplary embodiments being discussed below in detail.Each human genome has about 3 billion base pairs, requiring breaking thegenome into many parts and sequencing the parts in parallel to reducethe overall sequencing time and increase sequencing throughput. An arrayof individually addressable nanopores in conjunction with self-sealedchannels for DNA sequencing may significantly reduce the cost and timerequired for sequencing a human genome by allowing sequencing to beperformed in parallel by the various nanopores of the nanopore array.The self-sealed channels act as the fluidic reservoirs for the nanoporesin the nanopore array, and are formed by integrated circuit (IC)manufacturing methods using a sacrificial material without the need forwafer bonding. Because the self-sealed channels are relatively small,the physical size of the conductive fluidics containing the DNA are alsorelatively small, thus reducing parasitic capacitance between theelectrodes for high frequency applications.

Turning to FIG. 2, a flowchart illustrating an embodiment of a method200 of forming a nanopore array with self-sealed fluidic channels isshown. FIG. 2 is discussed with reference to FIGS. 3-12. Initially, asubstrate, or wafer, as is shown in FIG. 3, is provided. Substrate 300of FIG. 3 includes a back layer 301 and front layer 303, with a buriedlayer 302 located between the front layer 303 and the back layer 301.Front layer 301, buried layer 302, and back layer 303 may be anyappropriate solid state materials so long as front layer 301 and backlayer 303 are different materials from buried layer 302. Substrate 300may include a silicon-on-insulator (SOI) substrate in some embodiments,in which back layer 301 and front layer 303 are silicon, which may bedoped or undoped silicon in various embodiments, and buried layer 302 issilicon oxide. In other embodiments, back layer 301 and front layer 303may include any appropriate semiconductor materials, and may bedifferent materials, and buried layer 302 may include any appropriateinsulating material. In block 201 of FIG. 2, the front layer 303 ispatterned to form trenches, resulting in patterned front layer 401 withfront trenches 402 located between patterned front layer 401, as shownin FIG. 4. The patterned front layer 401 and front trenches 402 may beformed by etching into the front layer 303 and stopping on the buriedlayer 302; this may be accomplished, for example, by deep ultraviolet(DUV) photolithography and reactive ion etching (RIE).

As indicated in block 202 of FIG. 2, a membrane layer is deposited overthe patterned front layer 401 of FIG. 4. As more specificallyillustrated in FIG. 5, the membrane layer 501 is deposited overpatterned front layer 401 and in the front trenches 402 on the buriedlayer 302. Membrane layer 501 may be an insulating material including,but not limited to, silicon nitride in some embodiments, or may begraphene in other embodiments. The thickness of membrane layer 501 isselected such that nanopores may be formed in the membrane layer 501(discussed in further detail below with respect to block 206), and maybe about 200 nanometers thick or less. More specifically, a siliconnitride membrane layer 501 may be about 20 nm thick in some exemplaryembodiments, or membrane layer 501 comprising a single layer of graphenemay be about 0.335 nm thick. It should be noted that the membrane layer501 is selected to be a different material with respect to the buriedlayer 302.

After formation of the membrane layer 501, a mechanical support layer isformed over the membrane layer 501, as generally indicated in block 203of FIG. 2. As specifically shown in FIG. 6, mechanical support layer 601is deposited over membrane layer 501. Mechanical support layer 601 isrelatively thick as compared to membrane layer 501, and may include oneor multiple layers in various embodiments. For example, mechanicalsupport layer 601 may include a bottom layer of silicon oxide (that maybe about 200 nm thick, for example) located adjacent to membrane layer501, and a top layer of silicon nitride (that may also be about 200 nmthick, for example) located over the layer of silicon oxide. Thethickness of the mechanical supporting layer may vary from nanometers tomicrons depending on the application and the employed material(s). Forexample, formation of a relatively thick mechanical support layer 601may reduce ionic current noise in a nanopore sensor system duringoperation. While the mechanical support layer 601 may include anyappropriate insulating material in various embodiments, the portion ofmechanical support layer 601 that is adjacent to membrane layer 501 is amaterial that is different to the material that comprises membrane layer501 to allow selective etching of the mechanical support layer 601 whilestopping on the membrane layer 501.

Next, as indicated in block 204 of FIG. 2, windows are opened in themechanical support layer 601 corresponding to location at the bottoms offront trenches 402. As specifically shown in FIG. 7, the windows, suchas window 701, are etched into the mechanical support layer 601 toexpose the front side of membrane layer 501. Windows 701 are formed by,for example, wet and/or dry etching in various embodiments. Inembodiments in which mechanical support layer 601 includes a bottomlayer of silicon oxide located adjacent to membrane layer 501, and a toplayer of silicon nitride located over the layer of silicon oxide, thedry etch (such as carbon tetraflouride based reactive ion etching) mayfirst be used to open the top layer of silicon nitride, and then a wetetch (such as hydrofluoric etchant) may be used to open up the bottomlayer of silicon oxide; the wet etch stops when membrane layer 501 isreached.

Next, as indicated in block 205 of FIG. 2, the back layer 301 and buriedlayer 302 are patterned to form back trenches that are aligned withfront trenches 402. As specifically shown in FIG. 8, the back trenches803 are located between patterned back layer 801 and patterned buriedlayer 802, and expose the back side of membrane layer 501. Back trenches803 are aligned with front trenches 402. Back trenches 803 may be formedby etching into back layer 301 and stopping on the buried layer 302,which may be accomplished by DUV photolithography and RIE, therebydefining patterned back material 801. This exposes a portion of the backsurface of buried layer 302, which can be dry or wet etched to removethe exposed portion of the buried layer 302 on the back side of thesubstrate, in turn forming patterned buried layer 802. Back trenches 803expose the back side of membrane layer 501.

After formation of the back trenches 803, both the front and back sidesof membrane layer 501 are exposed, allowing formation of nanopores inmembrane layer 501, as indicated in block 206 of FIG. 2. As specificallyshown in FIG. 9, the nanopores 901 that are formed in membrane layer 501connect the front trenches 402 with the back trenches 803. The nanoporesmay be formed in membrane layer 501 using a focused electron beam from atransmission electron microscope (TEM) in some embodiments. The beamfrom a TEM is relatively small, for example on the order of a fewnanometers in diameter in some embodiments, allowing formation ofcommensurately small nanopores 901 in membrane layer 501. In otherembodiments, the nanopores 901 may be formed in membrane layer 501 byfocused ion beam sculpting or RIE with a hard mask. Nanopores 901typically have sizes ranging from sub-nanometer to tens of nanometers.

Then, as indicated in block 207 of FIG. 2, a sacrificial material isdeposited over the front and back of the structure of FIG. 9, includingthe front trenches 402, back trenches 803, and nanopores 901. FIG. 10shows the device of FIG. 9 after deposition of the sacrificial material1001. The sacrificial material 1001 may be patterned after deposition toexpose the top surface of the mechanical support layer 601. Sacrificialmaterial 1001 includes a thermally decomposable material, which mayinclude a polycarbonate in some embodiments (discussed in further detailbelow with respect to FIGS. 15 a-b). Sacrificial material 1001 may bedeposited by spin coating, for example.

Next, as indicated in block 208 of FIG. 2, front and back insulatinglayers are deposited over the front and back surfaces of the structureof FIG. 10. As specifically shown in FIG. 11, the front insulating layer1101 a covers the top surface of the sacrificial material 1001 and themechanical support layer 601 on the front side of the device, and theback insulating layer 1101 b covers the bottom portions of thesacrificial material 1001 and the patterned back layer 801 on the backside of the device. Front and back insulating layers 1101 a-b may besilicon nitride in some embodiments.

After formation of front and back insulating layers 1101 a-b, holescorresponding to channel endpoints (shown and discussed below withrespect to FIGS. 13 and 14) are formed in the front and back insulatinglayers 1101 a-b. Then, in block 209 of FIG. 2, the sacrificial material1001 is removed to form front and back channel pairs. FIG. 12 shows thedevice of FIG. 11 after removal of sacrificial material 1001 to formfront channels 1201 a and back channels 1201 b. Because the sacrificialmaterial 1001 is a thermally decomposable material, the sacrificialmaterial 1001 is removed by heating the device 1100 of FIG. 11 to atemperature at which the sacrificial material 1001 decomposes into gasphases, which escape through the holes corresponding to the channelendpoints in the front and back insulating layers 1101 a-b. Thesacrificial material removal temperature may be from about 200° C. toabout 400° C., depending on decomposition temperature of the sacrificialmaterial 1001, in various embodiments. Front channels 1201 a and backchannels 1201 b comprise self-sealed front and back channel pairs,wherein each pair of front and back channels is connected by a singlenanopore 901.

FIG. 13 is a schematic diagram illustrating a top view of an embodimentof a nanopore array with self-sealed fluidic channels formed by themethod of FIG. 2. With reference to FIG. 12, the top view shown in FIG.13 is looking down at the top insulating layer 1101 a of FIG. 12. Device1300 of FIG. 13 includes a plurality of self-sealed channels, such aschannel 1302, arranged on a substrate 1301. Each of the channels shownin FIG. 13 corresponds to a front channel 1201 a as shown in FIG. 12,and is connected via a single nanopore (shown in FIG. 14) to a backchannel (shown in FIG. 14). Holes 1303 and 1304 are formed on either endof channel 1302, in the top insulating layer 1101 a that forms the topsurface of the channel 1302. As discussed above with respect to block209 of FIG. 2, the sacrificial material 1001 escapes through holes 1303and 1304 after it is thermally decomposed. The holes 1303 and 1304 areconnected to external fluidic reservoirs 1306 and 1308 via tubes 1305and 1307. The fluidic reservoirs 1306 and 1308 hold a liquid, such as anionic buffer, that contains DNA to be analyzed by the nanoporeassociated with channel 1302. Fluidic reservoirs 1306 and 1308 maycomprise syringes in some embodiments. One of tubes 1305 and 1307 may bea fluidic outlet from channel 1302, and the other may be a fluidic inletto load the liquid into channel 1302, causing the liquid to flow fromone end of the channel 1302 to the other. The other channels shown onthe front side of substrate 1301 in FIG. 13 may also be connected torespective fluidic reservoirs and tubes, similarly to channel 1302, aswell as the back channels located on the other side of the substrate1301. Each channel shown on substrate 1301 is connected to a singlenanopore, and each nanopore is individually addressable and may be usedindividually for DNA sequencing, allowing sequencing to be performed inparallel by the nanopores in nanopore array 1300. FIG. 13 is shown forillustrative purposes only; a nanopore array with self-sealed fluidicchannels may include any appropriate number of nanopores with associatedfront and back channel pairs.

FIG. 14 shows a cross-section of channel 1302 of FIG. 13 along dashedline 1309 during a DNA translocation experiment. Holes 1403 and 1404 areopenings to the front channel, corresponding to holes 1303 and 1304 ofFIG. 13, tubes 1401 and 1402 correspond to tubes 1305 and 1307 of FIG.13, and fluidic reservoirs 1411 and 1412 correspond to fluidicreservoirs 1306 and 1308 of FIG. 13. Holes 1405 and 1406 are openingsinto the back channel. Tube 1407 connects fluidic reservoir 1409 to hole1405, and tube 1408 connects fluidic reservoir 1410 to hole 1406. Fluid1413, containing DNA molecules such as DNA molecule 1414, from fluidicsources 1409, 1410, 1411 and/or 1412 fills the front and back channels,and also fills the nanopore 901. Because DNA is negatively charged, thebattery configuration of electrical voltage bias 415 will cause the DNAto move from the top chamber to bottom chamber. So, in embodiments thatinclude a battery configuration such as electrical voltage bias 1415,DNA 1414 will be initially loaded on to top chamber only. During themeasurement, DNA 1414 will move from the top chamber to the bottomchamber through the nanopore 901. The DNA molecule 1414 is translocatedthrough nanopore 901 by the electrical voltage bias 1415 that is appliedacross the nanopore 901 via two electrochemical electrodes 1416 and1417, which are dipped in the fluid in fluidic reservoirs 1412 and 1410respectively. As the DNA molecule 1414 is translocated through nanopore901, the DNA molecule 1414 passes through the nanopore 901 base by base,allowing sequencing of the DNA 1414. FIG. 14 shows patterned back layer801, patterned buried layer 802, patterned front layer 401, membrane501, mechanical support layer 601, and front and back insulating layers1101 a-b. The front channel is bounded by front insulating layer 1101 a,and the back channel is bounded by back insulating layer 1101 b.

FIGS. 15 a-b show examples of thermally decomposable polymers that maybe used for sacrificial material 1001. FIG. 15 a shows polypropylenecarbonate (PPC) 1500 a, and FIG. 15 b shows polynorborene carbonate(PNC) 1500 b. Some PPC-based materials, such as shown in FIG. 15 a, maybe decomposed at a temperature from about 200 to about 350° C. SomePNC-based materials, such as shown in FIG. 15 b, may be decomposed at atemperature from about 250 to about 400° C. Both PPC and PNC basedmaterials are primarily converted into gaseous H₂O and CO₂ by thermaldecomposition.

The technical effects and benefits of exemplary embodiments includeformation of channels for a nanopore array that may be used for parallelDNA sequencing without wafer bonding.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an”, and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated.

1. A nanopore array, comprising: a plurality of front and back channelpairs located in a substrate, wherein the front channel of each channelpair is connected to the back channel of its respective channel pair bya single nanopore of a plurality of nanopores, wherein the nanopores areformed through a membrane layer that is located between the frontchannels and the back channels; wherein the plurality of front channelsof the plurality of front and back channel pairs are located in apatterned front layer of the substrate and bounded on top by a frontinsulating layer; wherein the membrane layer is located over thepatterned front layer and in a bottom portion of the plurality of frontchannels; and wherein the plurality of back channels of the plurality offront and back channel pairs are located in a patterned buried layer anda patterned back layer of the substrate and bounded at the bottom by aback insulating layer.
 2. The nanopore array of claim 1, wherein thefront and back layers comprise silicon, the buried layer comprises anoxide, the membrane layer comprises one of silicon nitride and graphene,and the front and back insulating layers comprise silicon nitride. 3.The nanopore array of claim 1, further comprising a mechanical supportlayer, the mechanical support layer being located over the membranelayer and in the front channels, the mechanical support layer comprisingwindows in which the nanopores are located.
 4. The nanopore array ofclaim 3, wherein the mechanical support layer comprises a first layer ofsilicon oxide formed adjacent to the membrane layer, and a second layerof silicon nitride formed over the first layer of silicon oxide.
 5. Thenanopore array of claim 4, wherein the first layer of silicon oxide hasa thickness of about 200 nm, and the second layer of silicon nitride hasa thickness of about 200 nm.
 6. The nanopore array of claim 1, whereinthe plurality of front and back channels are filled with a sacrificialmaterial comprising a thermally decomposable polymer.
 7. The nanoporearray of claim 6, wherein the sacrificial material comprises one ofpolypropylene carbonate (PPC) and polynorborene carbonate (PNC).
 8. Thenanopore array of claim 1, wherein the membrane layer is about 20 nmthick.